The biosynthesis of the endogenous beta-endorphin opioid peptide requires proteolytic processing of its POMC (proopiomelanocortin) precursor. Beta-endorphin is a key regulator of analgesia, behavior, and stress. It is, therefore, critical to define the proteolytic pathway(s) required to convert POMC into active beta- endorphin. Our studies have identified secretory vesicle cathepsin L as a key processing enzyme for POMC, based on exciting new results from cathepsin L knockout mice showing reduced levels of beta-endorphin. These novel results were obtained by purification from secretory vesicles, active-site affinity labeling, and peptide microsequencing to identify the processing activity as cathepsin L. Cathepsin L is localized to POMC-containing secretory vesicles, as well as neuropeptide-containing secretory vesicles. These new results implicate a significant role for secretory vesicle cathepsin L in beta-endorphin and neuropeptide production. The cleavage specificity of cathepsin L for dibasic processing sites generates peptide intermediates with NH2-terminal basic residues, indicating that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Arg/Lys aminopeptidase activity is colocalized in neurosecretory vesicles with beta-endorphin and neuropeptides. These new results indicate cathepsin L and Arg/Lys aminopeptidase as a new protease pathway for prohormone processing, in addition to the well known subtilisin-like PC1 and PC2 and carboxypeptidase E/H pathway. Thus, the goal of this proposal will be to determine the role of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, compared to PC1 and PC2, for processing POMC into beta- endorphin. This goal will be achieved in four specific aims to (1) determine the effects of reduced enzyme activities on POMC processing in (a) cathepsin L knockout mice, compared to PC1 and PC2 knockout mice, in pituitary and brain, (b) siRNA experiments to reduce enzyme levels in pituitary cells and brain neuronal cells, as well as in experiments for direct chemical inhibition of cathepsin L, (2) assess localization of cathepsin L with beta-endorphin and PC enzymes in secretory vesicles of pituitary and brain, (3) determine the role of each processing enzyme in (a) cellular POMC processing in PC12 neuroendocrine cells (and GH3 cells) by cotransfection of each enzyme with POMC, and in (b) in vitro kinetic and cleavage site studies of POMC processing, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for beta- endorphin production. Results will establish roles for secretory vesicle cathepsin L and Arg/Lys aminopeptidase processing pathway in the biosynthesis of beta-endorphin. New findings from this project will enhance our knowledge of the complexity of biosynthetic mechanisms for endogenous opioid and neuropeptide systems.